1. Field of the Invention
The invention concerns a method for the isolation and expansion of cardiac stem cells derived from postnatal cardiac tissue biopsy.
The invention deals with a method for the isolation, expansion and preservation of cardiac stem cells from human or animal tissue biopsy samples to be employed in cell transplantation and functional repair of the myocardium or other organs.
The cells may also be used in gene therapy, for treating genetic cardiomyopathies by expressing the healthy gene in cells from biopsies of subjects with genetic defects, propagating the cells in vitro and then transplanting them in the patient; for treating ischemic heart diseases by inducing the release of angiogenic growth factors by the transplanted cells; and for the setting of an in vitro models to study drugs.
2. Prior Art
Stem cells (SC) are able to replicate and to differentiate in response to appropriate signals, thus enabling the formation or regeneration of specialized tissues.
It was thought that cardiomyocytes were terminally differentiated cells; however, emerging evidence has shown the modest potential of these cells to proliferate in animal models and in heart transplant patients (1-4).
The limited ability of adult cardiomyocytes to undergo mitosis and to regenerate the myocardium after injury leads to a permanent deficiency in the number of functioning cells, with the development and progression of cardiac insufficiency. In the end stage of the disease, the alternative treatment to transplantation is the implantation of SC in the injured myocardium (cardiomyoplasty). This method has produced promising results in animal models and has been experimented also in humans. However, the problem of having a source and an availability of SC remains (5-7).
While embryonic SC (undifferentiated cells from the embryo that can produce a wide range of specialized cells and that can be derived from the cell mass inside blastocytes which, in humans, form 4-5 days after fertilization of the ovum) have a marked capability to proliferate and differentiate, their potential immunogenicity, arrhythmogenicity, and ethical issues in particular, have limited their use. Moreover, embryonic SC are pluripotent, consequently their use carries a potential risk of generating teratomas (as occurs in animal models). Hence, before these cells can be used, they need to be differentiated in vitro in cardiomyocytes.
There exist various types of cardiomyocytes (ventricular, atrial, sinus node, Purkinje, with pacemaker functions, etc.). Embryonic SC have the potential capability to generate these cardiomyocyte phenotypes in vitro but the yield is insufficient. Furthermore, the in vivo proliferative capability of cardiomyocytes derived from embryonic SC appears to be limited by the growth of multinucleate cells.
An alternative is to use adult SC (undifferentiated cells found in differentiated tissue that are able to proliferate, reproduce and differentiate into the specialized cell types of the tissues whence they were isolated) preferably obtained from the same patient, which would afford the advantage of allowing autologous transplantation without the need for immunosuppressive therapy. For this purpose, skeletal myoblasts (satellite cells) have been employed; however, they differentiate into skeletal myocytes with morphologic and functional properties differing from those of the cardiac muscle. The inability of skeletal myoblasts to transdifferentiate into cardiomyocytes and to couple with them could give rise to arrhythmias or other anomalies.
SC derived from bone marrow offer an attractive alternative, Mesenchymal SC (MSC) of the bone marrow can differentiate into cardiomyocytes in vitro (treated with DNA-demethylating agents) and in vivo where, however, in the presence of fibrosis, they mostly generate fibroblast-like cells. Hematopoietic SC (HSC) of the bone marrow (so-called side population cells [SPcells]) are pluripotent in that they can generate vascular epithelium, smooth muscle cells and cardiomyocytes. But the functional and electrophysiologic properties of HSC- and MSC-derived cardiomyocytes are not well characterized, and the use of undifferentiated cells instead of cardiomyocytes could give rise to in vivo differentiation into fibroblasts rather than muscle cells or to the development of tumors.
Although human cardiomyocytes have been conventionally considered terminally differentiated cells (i.e. unable to re-enter the cell cycle and to divide), indirect evidence accumulating over the past two years has suggested the existence of adult SC in the heart. These cells are ideal candidates for cardioplasty in that they need no reprogramming, give rise only to cells present in the heart, i.e. cardiomyocytes and vessels (endothelial cells and smooth muscles) and may, because this is their physiologic function, survive in transplant patients, integrate into the surrounding tissues and carry out their functions for longer periods without causing any damage. Patent applications WO 03/008535 and WO 03/006950 concern methods to derive cardiomyocytes from embryonic SC. Patent applications WO 02/13760 and WO 02/09650 deal with the use of adult SC (particularly hematopoietic and/or cardiac cells, without indicating a method to isolate them, also in combination) to repair cardiac injury or in treating cardiovascular diseases in general.
Patent application WO 99/49015 deals with the isolation of pluripotent cardiac SC of the adult p53−/− mouse. In particular, the description concerns the heart-derived pluripotent SC that differentiate and proliferate to produce a variety of cell types, including cardiocytes, fibroblasts, smooth muscle cells, skeletal muscle cells, keratinocytes, osteoblasts and chondrocytes. The cells may be employed in methods to treat patients with cardiac tissue necrosis. The SC proliferate and differentiate to produce cardiocytes that replace the necrotic tissue.
However, the method differs from that of the present invention, which was based on the assumption that the cardiac muscle cells, the striate muscles and the smooth muscle cells derived from a common precursor, the myoblast. Furthermore, there is no in vivo evidence from cardiomyopathic animals that supports the applicability of the method. Lastly, the methods differ substantially. In the method described in patent WO 99/49015, adult p53−/− mouse hearts are fragmented, dissociated with DNAse and collagenase. After centrifugation, the sediment myocytes are isolated on a discontinuous gradient (Percoll) and plated on a medium containing 5% FBS and then on a medium containing 15% FBS 20 days later. Between days 20 and 26, small (<5 μm) round, nonadherent, slow-growth, phase-bright cells with a high nucleus-to-cytoplasm ratio form in the suspension. These cells continue to live in the suspension for about 1.5 months in the presence of 10% horse serum. Then the cells remain suspended also without the addition of horse serum. The nonadherent SC do not form colonies in methylcellulose and proliferate in the presence of serum, SCF, aFGF, bFGF, and cFGF. In the absence of horse serum, the nonadherent cells differentiate into differently appearing adherent cells the authors have identified by mainly morphologic criteria as cardiocytes, chondrocytes, fibroblasts, smooth muscle cells, skeletal muscle myoblasts, pericytes, and other cells the authors have called adherent SC. About one fourth to one fifth of these cells is positive to alkaline phosphatase (osteoblasts and endothelial cells); all cells are negative to acetylated LDL (absence of endothelial cells) and to myosin heavy chain (MF20). The cells undergo mitosis when stimulated by bFGF, aFGF and cFGF. In the absence of serum, they differentiate into cells resembling a fried egg (myocytes), After treatment with ascorbic acid/α-GP, they differentiate into chondrocyte-like cells.
Adherent cells cloned by limiting dilution give rise to mesenchymal cells, including osteoblasts, chondrocytes, adipocytes and myocytes, although they cannot be clearly identified due to often inappropriate morphologic criteria and markers. All the cells tested negative to acetylated LDL (absence of endothelial cells). None of the 11 isolated clones could be induced to differentiate toward a single mesenchymal lineage.
The isolation of the cardiac-derived SC of neonate mice (1-4 days) is also described, wherein the passage of myocytes on human fibronectin is added to eliminate the fibroblasts. However, no data are given about the characteristics of the isolated SC. Furthermore, the cells isolated with the previous method do not give rise to the formation of an essential component of the heart tissues, i.e. vessels and endothelium.